Report on the 2014 Proficiency Test of the European Union Reference Laboratory for Mycotoxins, for the Network of National Reference Laboratories - Determination of Aflatoxin B1 in Copra (Coconut powder)

This report presents the results of the PT of the EURL for Mycotoxins which focused on the determination of aflatoxin B1 (Afla B1) in coconut powder samples. Sixty-one participants from 31 countries registered for the exercise and fifty-eight sets of results were reported. Only z-scores were used for an evaluation of performance. In total 91 % of the attributed z scores were below an absolute value of two, which indicated that most of the participants performed satisfactorily.JRC.D.5-Standards for Food Bioscienc

Report on the 2008 Proficiency Test of the Community Reference Laboratory for Mycotoxins, for the Network of National Reference Laboratories, regarding the Determination of Deoxynivalenol in a Cereal Product and a Test Solution

A proficiency test was conducted by the Community Reference Laboratory for Mycotoxins with 33 European National Reference Laboratories (NRLs) for Mycotoxins and 2 Laboratory from candidate countries, thus a total of 35 participants. Test materials were a deoxynivalenol (DON) solution in acetonitrile and three cereal test materials. Laboratories determined the DON content by either enzyme linked immuno sorbent assay (ELISA), gas chromatography (GC) or reverse-phase high-performance liquid-chromatography (RP-HPLC). One NRL did not report any results. Applying the Horwitz equation as a basis for the target standard deviation (19% in the case of this proficiency test), 27 out of the remaining 34 laboratories reported values within the z-score limit of 2 after recovery correction of the result for the DON-positive sample. Twenty-five laboratories reported results within a z-score limit of 1. Thus, 79 % of the participating laboratories performed satisfactorily in the proficiency test. No z scores were calculated for the blank material.JRC.D.8-Food safety and qualit

Report on the 2007 Proficiency Test for the Determination of Ochratoxin A in Capsicum ssp (Paprika Powder)

A proficiency test was conducted with 68 laboratories from 17 EU Member States and four Third Countries. Test materials were one naturally contaminated "Ochratoxin A positive" and one "Ochratoxin A blank" capsicum material. The majority of laboratories chose to determine the ochratoxin A content by reverse-phase high-performance liquid-chromatography (RP-HPLC) with fluorescence detection against their own standard solutions as reference. Applying the modified Horwitz equation according to Thompson as a basis for the target standard deviation (22% in the case of this proficiency test), 79% of the laboratories achieved z scores of less than ¦2¦. The results were evaluated further on the basis of the returned questionnaire that each participant received. The questions asked were focussed on the fact that future method development, if necessary, could be supported by comparison of the methodologies and method procedures applied.JRC.D.8-Food safety and qualit

Equivalence testing of histamine methods - Final Report

Histamine fish poisoning is an allergy-like form of food poisoning that continues to be a major problem in seafood safety. The FAO/WHO Codex Alimentarius as well as EU legislation have therefore set maximum limits for histamine in fish and fish products. The analytical methods requested by Codex and by EU are different and concern has been raised that this could lead to disputes in the international trade of seafood. This report describes the outcome of a study, commissioned by DG Health and Consumers and carried out by DG Joint Research Centre, that compared the performance of the method for determining histamine in fish as mandated by Regulation (EC) No 2073/2005 to the method mandated in Codex Alimentarius Standard 165-1989. The EU mandated method is based on HPLC separation of histamine and subsequent detection by a UV detector. It was published in the Journal of AOAC International, but has not been validated by a collaborative study. The Codex method is AOAC 977.13, which is a based on the formation of a fluorescent derivative of histamine and subsequent measurement in a fluorimeter; it has been validated by collaborative trial. The correct implementation of both methods by JRC was assessed by carrying out performance verification studies using various canned and fresh scromboid fish samples (tuna, macrel, and herring) taken from the Belgian market. Repeatability (RSDr) and intermediate precision (RSDip) as well as recovery data were generated. Both methods conformed to specifications. Various approaches were followed to test the equivalency of both methods, which were based on statistical hypothesis testing (t-test), regression analysis and benchmarking against established reference values. All approaches indicated that the two methods are not fully equivalent. The EU mandated method has a tendency to overestimate, while the Codex method has a tendency to underestimate the histamine content in fish. It was recognised that the EU mandated method was very accurate when applied to fresh tuna. A distinct matrix influence was noticed for all other fish species tested, leading to an overestimation of the histamine content. It is therefore recommended to optimise the EU method so that matrix effects can be eliminated, or at least taken into account in an appropriate manner, In addition, a collaborative trial for the HPLC method to establish reproducibility data for the method should be organised. In line with current practice the collaborative study should also require to correct the reported data for recovery. Furthermore, as an ad-hoc measure the replacement of the HPLC method mentioned in Regulation (EC) No 2073/2005 by a ring-tested HPLC method, which are already available, could be considered.JRC.D.5-Standards for Food Bioscienc

Report on the 2012 Proficiency Test on pyrrolizidine alkaloids in honey and hay

The purpose of this proficiency test was to investigate the current measurement capacities of testing laboratories for pyrrolizidine alkaloids in honey and plant materials. The scheme consisted of two parts: Benchmarking performance of laboratories against known estimates of pyrrolizidine alkaloids in the samples and checking for methodological differences while measuring naturally contaminated materials. Twenty-eight laboratories expressed their will to participate and analysed multiple analytes in several test samples of honey and plant material. The analysis of spiked honey showed no statistical differences between determining a common sum parameter for alkaloids and individual determination. A significant difference has been found however for of naturally contaminated materials. Individual alkaloid determination showed significantly lower results, possibly because of the presence of substances contributing to the sum parameter that were not in the scopes of the methods applied as well as lack of standard materials available on the market. Satisfactory performance for all of analytes has been achieved by more than half of participants analysing for both: sum parameter, and alkaloids analysed individually.JRC.D.5-Standards for Food Bioscienc

Report on the 2017 Proficiency Test of the European Union Reference Laboratory for Mycotoxins: determination of ergot alkaloids in rye

Throughout history, there have been several deadly episodes of food poisoning by ergot alkaloids (EAs) (known as St. Anthony’s fire or ergotism). EAs are secondary metabolites produced by fungi of the Claviceps genus (chiefly Claviceps purpurea), which are common pathogens of cereals and pasture grasses. During harvest, the fungal body is collected together with the crop leading to the contamination of cereal based food and feed products. Although this event is highly attenuated nowadays by the physical cleaning techniques in the mills, the detection of EAs in food and feed commodities is not infrequent. Since 2002, the EU legislation (Directive 2002/32/EC) sets up the maximum content for rye ergot (sclerotia) in all feed containing unground cereals. However, the visual determination of sclerotia in cereals is often inaccurate. Moreover, this visual determination is impossible in processed food and feed. Additionally, the pattern of EAs levels in relation to fungal strains, geographical distribution and host plant is not fully known and they cannot be directly related to the sclerotia amount visually determined. The Commission Recommendation 2012/154/EU additionally recommends the monitoring of the presence of individual EAs in feed and food by chemical analytical methods. A proficiency test (PT) was organised by the European Union Reference Laboratory (EURL) for Mycotoxins targeting the determination of the most prominent EAs in Claviceps purpurea: ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine and their related –inine epimers, as listed in the above Recommendation. The levels in the rye test material varied from 116 (ergometrine/inine) to 752 µg/kg (ergocristine/inine). Thirty-seven laboratories, among them 26 National Reference Laboratories for mycotoxins in food and feed from 21 EU Member States plus Iceland and Norway, and 11 Official Control Laboratories participated in the PT. The rating of the laboratories' performance was done by means of z-scores considering the sum of the -ine/-inine pairs of epimers with respect to the values assigned at the JRC-Geel and a pt of 22 %. Ninety one percent of the results were classified as satisfactory (|z| ≤ 2), while 3.7 % fell into the unsatisfactory range (|z| ≥ 3). All the results received for ergocornine/inine were satisfactory, whereas 76 and 86 % of the results for -ergocryptine/inine and ergometrine/inine, respectively, were classified as satisfactory. Despite the overall good performance of the laboratories analysing EAs in the test item, this PT highlighted the need to seek a harmonised approach for quantifying a- and β-ergocryptine/inine, as currently only the a-isomers of this EA type are available as pure reference materials.JRC.F.5-Food and Feed Complianc

Validation of an Analytical Method to Determine the Content of Fumonisins in Baby Food, Breakfast Cereals and Animal Feed

An inter-laboratory comparison was carried out to evaluate the effectiveness of a method based on immunoaffinity column clean-up followed by derivatisation and high performance liquid chromatography with fluorimetric quantification (HPLC-FL). The method was tested for the determination of Fumonisins B1 and B2 (FB1 & FB2) in baby food, breakfast cereals and animal feed to monitor compliance with limits according to Regulation 1881/2006/EC and Recommendation 576/2006/EC. The test portion of the sample was extracted with methanol:water. The sample extract was filtered, diluted, passed over an immunoaffinity column for clean-up and evaporated. The redissolved and purified eluate was separated and determined by reverse-phase high performance liquid chromatography (HPLC) and fluorescence detection after the fumonisins had been derivatised to their fluorescent isoindols in the presence of o-phthaldehyde and a thiol-coupling reagent with either pre- or post-column derivatisation. Baby food, breakfast cereal and animal feed samples, both blank and naturally contaminated with FB1 and FB2, were sent to 40 laboratories from 19 EU Member States, and a laboratory in Uruguay. For recovery determination extra test portions of the blank samples were to be spiked by the participants at levels of 135 µg/kg for the sum of FB1 and FB2 in baby food, 400 µg/kg in breakfast cereals, and 3700 µg/kg in animal feed. All samples were sent as blinded duplicates. Mean recoveries were calculated as 71 % for baby food, and 87 % for breakfast cereals. Based on results for the spiked and naturally contaminated samples the relative standard deviations for reproducibility (RSDR) in baby food were 31 % at a spiked level of 135 µg/kg, 44 % at a natural contamination level of 267 µg/kg, and 33 % at a natural contamination level of 501 µg/kg. For breakfast cereal these figures were 15 % at a spiked level of 400 µg/kg, and 33 % at a natural contamination level of 1034 µg/kg. The values for RSDr in those materials ranged from 5 to 29 % in baby food and 12 to 14 % in breakfast cereal.JRC.DG.D.6-Food Safety and Qualit

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the network of National Reference Laboratories: Determination of aflatoxin B1 in defatted peanut powder

The Joint Research Centre (JRC), a Directorate-General of the European Commission, operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise proficiency tests (PTs) among appointed National Reference Laboratories (NRLs). This report presents the results of the PT on the determination of aflatoxins in defatted peanut powder. The test items for this PT were two contaminated defatted peanut powder samples. The materials were produced by the JRC and dispatched to the participants mid October 2016. Each participant received one bottle per test material containing approximately 55 g each. Fifty-six participants from thirty countries (among them 40 NRLs and 16 official food control laboratories) registered for the exercise and 54 sets (Sample A and B) of results were reported. The assigned values, established by an exact-matching double isotope dilution mass spectrometric technique, were 2.80 µg/kg (± 0.19 µg/kg) aflatoxin B1 in sample A and 3.20 µg/kg (± 0.20 µg/kg) in sample B. Participants' results were rated with z-scores and zeta-scores for aflatoxin B1 in accordance with ISO 13528:2015. The z-score compares the participant's deviation from the reference value with the target standard deviation accepted for the PT, whereas the zeta-score provides an indication of whether the participant's estimate of uncertainty is consistent with the observed deviation from the assigned value. Only z-scores were used for the evaluation whether an individual laboratory underperformed. In total, 96 % of the attributed z scores were below an absolute value of two for sample A and and 92 % for sample B. This indicates that most of the participants performed satisfactorily. The few participants that had z-scores above an absolute value of 2 will have to investigate the reasons for the deviation (root-cause analysis) and report the planned corrective actions to the EURL.JRC.F.5-Food and Feed Complianc

Validation of an Analytical Method to Determine the Content of Ochratoxin A in Animal Feed

An inter-laboratory comparison was carried out to evaluate the effectiveness of a method based on immunoaffinity column clean-up followed by high performance liquid chromatography using fluorimetric detection (HPLC-FL). The method was tested for the determination of ochratoxin A (OTA) in animal feed at concentration levels relevant to those proposed according to Commission Recommendation 2006/576/EC ( ). The test portion of the sample was extracted with methanol:water. The sample extract was filtered, diluted, passed over an immunoaffinity column for clean-up and then eluted with methanol. The separation and determination of the OTA was performed by reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection with an excitation of 333 nm and emission of 467 nm. The animal feed samples, both spiked and naturally contaminated with OTA, were sent to 35 laboratories in 15 EU Member States, Colombia, Canada and Japan. Each laboratory received 6 duplicate samples ¿ 4 coded and 2 blanks for spiking with coded solutions. Blank marked test portions of the samples were spiked at levels of 76 µg/kg and 305 µg/kg OTA. The range of recovery values reported spanned from 47 % ¿ 124 % with an average value of 82 % and 79 % for each level respectively. Based on results for spiked samples (blind duplicates at two levels), as well as naturally contaminated samples (blind duplicates at three levels), the relative standard deviation for repeatability (RSDr) ranged from 3.1 % ¿ 4.7 %. The relative standard deviation for reproducibility (RSDR) ranged from 13.5 % ¿ 14.6 %. After correction for recovery, the RSDR values improved significantly and ranged from 5.6 % ¿ 6.4 %for naturally contaminated test materials. This method therefore showed acceptable within-laboratory and between-laboratory precision for each matrix.JRC.D.8-Food safety and qualit

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in tea and herbal infusions

Tropane alkaloids (TAs) are toxins found in a wide variety of plant species growing in mild climates. The most well-known are Datura, Atropa and Hyoscyamus sp., belonging to the Solanaceae family. The TAs family comprises more than 200 compounds, of which atropine and scopolamine are the most active producing anticholinergic symptoms (e.g. blurred vision, dry mouth, muscle spasms, tachycardia and death) if ingested in toxic quantities. The presence of botanical impurities (e.g. seeds, leaves and roots) has been reported in a variety of tea and herbal blends, stressing the need to control the quality of these products in the EU market. The European Union Reference Laboratory (EURL) for Mycotoxins organised a proficiency test (PT) on the determination of TAs (atropine and scopolamine) in tea and herbal infusions upon request from DG SANTE. The measurand levels were targeted to provide insight on the measurement capabilities of EU Member States' laboratories at concentrations close to the recommended limit of quantification (LOQ) established by the Commission Recommendation 2015/976 (preferably below 5 μg/kg and not higher 10 μg/kg). Additionally, the ratio of atropine to scopolamine was kept as native in the plant materials in some samples. Three matrices appropriately processed were provided to the participants: black tea, peppermint leaves and fennel seeds. The concentrations of atropine varied from 8.3 to 42.2 μg/kg while those of scopolamine ranged from 1.5 to 20.8 μg/kg. The participants were asked to determine atropine and scopolamine in 6 contaminated samples (2 per matrix) and 3 blank materials spiked by them with a TAs solution of unknown concentration. This setup was also aimed to allow a preliminary assessment of the robustness of the EURL-developed method. Thirty-three laboratories from 11 Member States joined the PT, with a very significant participation from Germany. The performance of the laboratories was assessed using z-scores with regard to the assigned values obtained by exact matching double isotope dilution mass spectrometry (EMD-IDMS), in line with the ISO 13528:2015. In all cases, the consensus values derived from the participants' data were within the range of the assigned values, considering the respective confidence intervals. On average, eighty-seven percent of the z-scores for atropine and 84 % for scopolamine fell in the acceptable range (|z| ≤ 2). The success rate varied from 83 to 94 % for atropine and from 67 to 94 % for scopolamine, across the distributed matrices and concentration levels. The robust standard deviations of the reported results for both TAs were in good agreement with the target standard deviation (22 %). The results of this PT indicate that EU Member States’ laboratories can determine atropine and scopolamine reliably in tea and herbal infusions at levels relevant to the current legislation (Commission Recommendation 2015/976).JRC.F.5-Food and Feed Complianc